Acute kidney injury (AKI) carries high morbidity and mortality. The only treatment that is currently available for AKI consists of supportive measures. The overall goal of this project is to develop stem cell- based therapy to treat AKI. We have previously shown that freshly isolated mouse hematopoietic stem cells (HSC) can be transplanted and incorporated into regenerating renal tubules. However, the incorporation rate is low and no functional benefit is observed. To enhance the therapeutic potential of HSC, we have developed a novel approach in which hematopoietic stem and progenitor cells are treated sequentially with cytokines, nephrogenic factors, and an HDAC inhibitor trichostatin A to induce renal differentiation prior to transplantation. Induced cells have de novo expression of renal developmental genes and down-regulation of hematopoietic differentiation genes. Injection of induced cells or induced cell-conditioned medium improves renal function in mice with AKI, suggesting renal protection by endocrine/paracrine effects. Furthermore, blocking the calcium sensing receptor, CaR, on induced cells increases renal localization of the cells. This result suggests that modulation of the CaR may be useful to increase intrarenal delivery of the cells to achieve greater therapeutic effects. To extend these promising findings, we propose to characterize induced cells with additional renal markers to confirm the selection of renal cell fate. We will determine whether induced cells can differentiate into functional renal epithelial cells by in vitro assays and transplantation studies. The molecular pathways involved in cell conversion will be analyzed to guide the discovery of optimal conditions for cell conversion (Aim 1). We will examine whether induced cells exert endocrine/paracrine effects by decreasing death and increasing proliferation of endogenous tubular epithelial cells and/or endothelial cells. Since surviving tubular cells are the main source for renal repair, renotrophic factors released by induced cells can be important agents to treat AKI (Aim 2). Furthermore, we will block the CaR with an antibody or use CaR-deficient mice to test whether modulation of the CaR could increase intrarenal transmigration of the cells. This strategy may increase the therapeutic utility of induced cells by increasing tubular integration for direct cell replacement and providing more sustained delivery of renotrophic factors to the injured tubules (Aim 3). In conclusion, we take novel approaches to develop cell therapeutic agents to treat AKI by sequential treatment of hematopoietic stem and progenitor cells that can be obtained easily for donor-directed therapy. PUBLIC HEALTH RELEVANCE: Narrative Acute kidney injury has high morbidity and high mortality. There is no specific and effective treatment at present time. Stem cells offer therapeutic potential for kidney disease. The goal of this application is to develop stem cell-based therapy to treat acute kidney injury.